This project has two primary aims. The first aim is to understand how proteins which are secreted or inserted into membranes are targeted to transport sites in the endoplasmic reticulum (ER) or, equivalently, the bacterial inner membrane (IM). In particular we are investigating the role of a ribonucleoprotein called the signal recognition particle (SRP) and its ER-bound receptor in this process. Previous studies have shown that SRP recognizes nascent polypeptide chains containing signal sequences and then catalyzes their translocation across the ER membrane upon interaction with the SRP receptor. We plan to elucidate the mechanism by which SRP recognizes the highly diverse family of signal sequences found on different proteins and then releases them at the surface of the ER in a regulated fashion. By studying this problem we expect to obtain insight into the function and regulation of proteins that possess broad substrate specificity. The second aim of the project is to elucidate the function of a universal protein conducting channel or translocon that is found in both the ER and the IM. We are particularly interested in understanding how the translocon catalyzes two related but seemingly distinct processes, namely the complete transfer of presecretory proteins across a membrane and the integration of membrane proteins into a lipid bilayer. In addition, we would like to determine why the core of the translocon is evolutionarily conserved, but the peripheral subunits are not. We expect that these experiments will provide significant insight into the function of membrane-bound channels. In recent work we have focused on the function of E. coli homologs of SRP, the SRP receptor and the translocon using a combination of biochemical and genetic methods. The function of SRP in bacteria has been of particular interest in light of substantial evidence that bacteria utilize predominantly chaperone-based mechanisms to target secreted proteins to the IM. We have found that E. coli SRP targets only inner membrane proteins (IMPs) to the IM, and therefore has a much more restricted function than its mammalian counterpart. Interestingly, we have found that some IMPs, in particular a subset of bitopic IMPs, do not require SRP for their insertion into the IM. Domain swapping experiments and deletion analysis showed that SRP- dependence correlates with the presence of specific cytoplasmic or periplasmic domains which presumably promote misfolding of the protein in the absence of SRP. Further experiments also showed that the insertion of many polytopic IMPs is only partially SRP-dependent. Considered together, the data suggest that in the absence of SRP there would be a competition between proper insertion of IMPs and aggregation in the cytoplasm and that SRP is required primarily to accelerate targeting and to push the equilibrium towards effective insertion. Finally, we have obtained strong evidence that the SecA protein, a peripheral membrane protein that has previously been shown to be required only for the post-translational insertion of secreted proteins, is also required for the insertion of IMPs that are targeted by SRP. This finding was unexpected in light of previous studies and suggests that there are significant differences in the protein translocation process in eukaryotic and prokaryotic cells. - protein translocation, protein targeting, ribonucleoprotein, signal sequence, membranes, secretion, endoplasmic reticulum